Peptide compounds to regulate the complement system

ABSTRACT

The present invention provides peptide compounds that regulate the complement system and methods of using these compounds. The invention is an isolated, purified peptide of 30 amino acids derived from human astrovirus protein, called CP1. The invention is directed to peptide compounds that are peptide mimetics, peptide analogs and/or synthetic derivatives of CP1 having, for example, internal peptide deletions and substitutions, deletions and substitutions at the N-terminus and C-terminus, and that are able to regulate complement activation. The invention further provides pharmaceutical compositions of therapeutically effective amounts of the peptide compounds and a pharmaceutically acceptable carrier, diluent, or excipient for treating a disease or condition associated with complement-mediated tissue damage.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 15/203,469,entitled “Peptide Compounds to Regulate the Complement System,” filed onJul. 6, 2016, which is a continuation of U.S. application Ser. No.14/536,073, entitled “Peptide Compounds to Regulate the ComplementSystem,” filed Nov. 7, 2014 now U.S. Pat. No. 9,422,337 issued Aug. 23,2016, which is a division of U.S. application Ser. No. 13/809,371,entitled “Peptide Compounds to Regulate the Complement System,” filedMay 23, 2013 now U.S. Pat. No. 8,906,845 issued Dec. 9, 2014, which is a§ 371 of PCT/US2011/044791 entitled “Peptide Compounds to Regulate theComplement System,” filed Jul. 21, 2011, which claims priority to U.S.Provisional Application No. 61/366,204 entitled “Peptide Compounds toRegulate the Complement System,” filed Jul. 21, 2010, all of which areincorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant R21 AI060874awarded by National Institutes of Health (NIH). The government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 25, 2013, isnamed 0113019.270US1_SL.txt and is 13,348 bytes in size.

FIELD

The invention relates generally to the field of therapeutic interventionin inflammatory and autoimmune disease. More specifically, the inventionrelates to peptide compounds that can regulate complement activation andcan be used therapeutically in the prevention and treatment ofcomplement-mediated diseases, such as inflammatory, autoimmune andpathogenic diseases.

BACKGROUND

The complement system, an essential component of the innate immunesystem, plays a critical role as a defense mechanism against invadingpathogens, primes adaptive immune responses, and helps remove immunecomplexes and apoptotic cells. Three different pathways comprise thecomplement system: the classical pathway, the lectin pathway andalternative pathway. C1q and mannose-binding lectin (MBL) are thestructurally related recognition molecules of the classical and lectinpathways, respectively. Whereas IgM or clustered IgG serve as theprincipal ligands for C1q, MBL recognizes polysaccharides such asmannan. Ligand binding by C1q and MBL results in the sequentialactivation of C4 and C2 to form the classical and lectin pathwayC3-convertase. In contrast, alternative pathway activation does notrequire a recognition molecule, but can amplify C3 activation initiatedby the classical or lectin pathways. Activation of any of these threepathways results in the formation of inflammatory mediators (C3 and C5a)and the membrane attack complex (MAC), which causes cellular lysis.

While the complement system plays a critical role in many protectiveimmune functions, complement activation is a significant mediator oftissue damage in a wide range of autoimmune and inflammatory diseaseprocesses. (Ricklin and Lambris, 2007).

A need exists for complement regulators. While the complement system isa vital host defense against pathogenic organisms, its uncheckedactivation can cause devastating host cell damage. Currently, despitethe known morbidity and mortality associated with complementdysregulation in many disease processes, including autoimmune diseasessuch as systemic lupus erythematosus, myasthenia gravis, and multiplesclerosis, only two anti-complement therapies have recently beenapproved for use in humans: purified, human C1-Inhibitor licensed foruse in patients suffering from hereditary angioedema (HAE) andEculizumab/Solaris, a humanized, long-acting monoclonal antibody againstC5 used in the treatment of paroxysmal nocturnal hemoglobinuria (PNH)Both PNH and HAE are orphan diseases in which very few people areafflicted; currently no complement regulators are approved for the morecommon disease processes in which dysregulated complement activationplays a pivotal role.

The Astroviridae constitute a family of non-enveloped, icosahedralviruses with a single-stranded, messenger-sense RNA genome. Theseviruses are a significant cause of gastroenteritis in humans as well asother diseases in other animal species. It is estimated that they causean estimated 2-17% of children's diarrheal illness worldwide.

The astrovirus coat protein (“CP”) has strong effects on the complementsystem, suggesting that the ‘active’ portion of the protein may haveclinical utility in decreasing tissue damage from complement-mediateddiseases. The wild type coat protein (“WP CP”) purified from humanastrovirus type 1 (HAstV-1) can bind C1q and MBL, and regulates bothclassical and lectin pathway activations (Hair et al., 2010. Molec.Immunol. 47, 792-798). This property is analogous to the propertiesdescribed for human neutrophil peptide-1 (HNP-1) (van den Berg et al.,1998. Blood. 92, 3898-3903; Groeneveld et al., 2007. Molec. Immunol. 44,3608-3614). The HAstV-1 coat protein is a 787 amino acid molecule thathas been expressed from a recombinant baculovirus construct and thenpurified (Bonaparte et al., J. Virol. 82, 817-827).

Developing peptide compounds to inhibit classical, lectin andalternative pathways of the complement system are of interest, as eachof these three pathways have been demonstrated to contribute to numerousautoimmune and inflammatory disease processes. Specific blockade ofclassical and lectin pathways are of particular interest, as both ofthese pathways have been implicated in ischemia-reperfusion inducedinjury in many animal models. (Castellano et al., 2010; Lee et al.,2010; Tjernberg, et al., 2008; Zhang et al. 2006). Humans withalternative pathway deficiencies suffer sever bacterial infections;thus, a functional alternative pathway is essential for immunesurveillance against invading pathogens.

It would be desirable to develop peptide compounds that can regulatecomplement activation and can be used therapeutically to prevent andtreat complement-mediated diseases, such as inflammatory, autoimmune andpathogenic diseases.

SUMMARY

The present invention provides peptide compounds that regulate theclassical and lectin pathways of the complement system and methods ofusing these compounds. Specifically, the peptide compounds of thisinvention can bind, regulate and inactivate C1 and MBL, and thereforecan efficiently inhibit classical and lectin pathway activation at itsearliest point while leaving the alternative pathway intact. Thesepeptide compounds are of therapeutic value for selectively regulatingand inhibiting C1 and MBL activation without affecting the alterativepathway and can be used for treating diseases mediated by dysregulatedactivation of the classical and lectin pathways. In other embodiments,the peptide compounds regulate the classical pathway activation but notthe lectin pathway activation.

The invention is based on the identification of an isolated, purifiedpeptide of 30 amino acids derived from human astrovirus coat protein,termed CP1, and having SEQ ID NO:1 that is able to regulate theclassical and lectin pathway activation by binding to C1q and MBL.

In other embodiments, the invention is directed to peptide compoundsthat are peptide mimetics, peptide analogs and/or synthetic derivativesof CP1 having, for example, internal peptide deletions andsubstitutions, deletions and substitutions at the N-terminus andC-terminus, and that are able to regulate the classical and lectinpathway activation by binding to C1q and MBL.

A further embodiment of the invention is any one of the peptidecompounds of this invention, wherein the peptide compound is modifiedthrough acetylation of the N-terminal residue.

In some embodiments, the peptide sequence has at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity to SEQID NO:1.

Another embodiment of the invention further provides pharmaceuticalcompositions. For example, the invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of the peptideof any one of the compounds described above and at least onepharmaceutically acceptable carrier, diluent, or excipient.

Another embodiment of the invention further provides a method ofregulating the complement system in a subject, comprising administeringto the subject the composition described above.

A further embodiment of the invention is a method of treating a diseaseassociated with complement-mediated tissue damage by administering thepharmaceutical compositions described above, wherein the diseaseassociated with complement-mediated tissue damage is selected from thegroup consisting of rheumatoid arthritis, systemic lupus erythematosus,multiple sclerosis, myasthenia gravis, autoimmune hemolytic anemia,membranoproliferative glomerulonephritis, serum sickness, AdultRespiratory Distress Syndrome (ASDS), ischemia-reperfusion injury,stroke, myocardial infarction, allo- or xeno-transplantation injury,hyperacute rejection, graft versus host disease (GVHD), Alzheimer'sdisease, burn injuries, hemodialysis damage, cardiopulmonary bypassdamage, paroxysmal nocturnal hemoglobinuria (PNH), and hereditaryangioedema (HAE).

Another embodiment of the invention is a method of treating a diseaseassociated with complement-mediated tissue damage, further comprisingadministering to a subject at least one other active ingredienteffective in treating the disease, wherein the at least one other activeingredient is selected from the group consisting of a non-steroidalanti-inflammatory agent, a corticosteroid, a disease modifyinganti-rheumatic drug, a C1-inhibitor, and eculizumab.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Other features and advantages of the invention will be apparent from thedetailed description, drawings, and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting CP dose-dependently competing with humanneutrophil defensin 1 (HNP-1) for binding to C1q. C1q was mixed withincreasing amounts of WT CP (circles) or BSA (triangles) and added tothe ELISA plate coated with HNP-1. After washing, bound C1q was measuredusing polyclonal antibody to C1q. Data are the means from threeindependent experiments. Error bars denote SEM.

FIG. 2A shows the alignment of WT CP with the 30 amino acid HNP-1molecule as determined by ClustalW analysis. The symbol “*” indicatesidentical residues, “:” indicates conserved residues, and “.” indicatessemi-conserved residues between CP and HNP-1 sequences. FIG. 2A alsoshows the two 30 amino acid peptides (CP1 and CP2) that were synthesizedbased upon this alignment. FIG. 2B is a graph depicting peptidecompounds that dose-dependently competed with WT CP for C1q binding. Aconstant amount of C1q was mixed with increasing amounts of WT CP andadded to an ELISA plate coated with CP1 (no symbol) or CP2 (squares).When BSA was substituted for WT CP (triangles), no competition occurred.Data are the means from three independent experiments. Error bars denoteSEM.

FIGS. 3A-3D demonstrates the binding of CP peptides to C1q. PeptidesCP1, CP2, HNP-1 (FIG. 3A), C04A, C27A (FIG. 3B), E23A, E25A (FIG. 3C)and Δ8-22 (FIG. 3D) were coated onto the ELISA plate and incubated withincreasing amounts of purified C1q. C1q was detected with polyclonalantisera to C1q. Data represents triplicate readings for each peptidederivative. Error bars denote SEM.

FIGS. 4A-4D demonstrate that CP1, but not CP2, regulates C1 activation.Partially purified human C1 was incubated alone, with aggregated IgG(agg-IgG), or with agg-IgG and increasing amounts of CP1 (FIG. 4A) orCP2 (FIG. 4B) peptides (1-4 μl of a 250 mM stock) for 90 minutes at 37°C. The reaction mixtures were then loaded on an 8% SDS-PAGE gel andsubjected to immunoblot with polyclonal antisera to C1s. In FIGS. 4A and4B, the heavy and light chains of C1s, which indicate C1s activation,and the proenzyme C1s, are indicated to the right of the gel image,while the molecular mass markers (in kD) are indicated to the left ofthe gel image. FIGS. 4C and 4D are graphs quantifying the extent of C1activation corresponding to CP1 (FIG. 4C) and CP2 (FIG. 4D),respectively, as determined by Odyssey imaging. Data are the means fromtwo independent experiments. Error bars denote SEM.

FIG. 5 is a graph depicting peptide compound regulation of complementactivity in a C4 activation assay. ELISA plates were pre-coated withovalbumin decorated with anti-ovalbumin antibodies. NHS was incubatedalone or with BSA, dimethyl sulfoxide (DMSO) control, WT CP (1.8 μg), orpeptide compounds (0.5 mM) for 15 minutes and subsequently added to theovalbumin-antibody target. Polyclonal C4 antibody was used to detect C4deposition. C4 deposition was standardized to 100% for NHS alone, andall values were adjusted to subtract out background values from aheat-inactivated NHS control. Data are the means from three independentexperiments. Error bars denote SEM.

FIGS. 6A and 6B are graphs depicting peptide compound regulation ofcomplement activity in a hemolytic assay. In FIG. 6A,antibody-sensitized sheep erythrocytes were incubated with NHS alone, orwith peptide compounds (1.4 mM) or a DMSO control. In FIG. 6B,antibody-sensitized sheep erythrocytes were incubated with NHS (whitebars) or factor B-depleted serum (black bars) alone, or with peptidecompounds (0.77 mM) or a DMSO control. Hemolysis was standardized to100% for serum alone. FIG. 6A depicts the mean data from threeindependent experiments, and FIG. 6B depicts data from one experiment.Error bars denote SEM.

FIG. 7 is a graph depicting the hemolytic assay titration of the PolarAssortant peptide in Factor B-depleted serum. The data shows the PolarAssortant peptide regulating classical pathway activation in a dosedependent manner.

FIGS. 8A and B are graphs depicting MALDI-TOF-TOF mass spectrometryanalysis of the oligomeric state of E23A. FIG. 8A depicts linear modeanalysis of E23A, and FIG. 8B depicts reflection mode analysis of E23A.E23A has a theoretical mass of 2934.37. In FIG. 8A, the lower resolutionand lower mass accuracy of the linear mode is shown in the zoom of thepeptide peak with the lack of monoisotopic peptide peaks. In FIG. 8B,the high resolution and mass accuracy of the reflection mode is shown inthe zoom of the peptide peak. In both FIGS. 8A and 8B, there are nomajor peaks with a mass to charge ratio (m/z) greater than thetheoretical mass of the peptide.

FIG. 9A depicts the amino acid residues of E23A compared to the residuesof Vigna radiata plant defensin 1 (VrD1). E23A was uploaded ontoCPHModels-3.0 server, which aligned residues 2-29 of E23A with residues17-44 of the plant defensin VrD1. The alignment was confirmed byClustalW analysis. The symbol “*” indicates identical residues, “:”indicates conserved residues, and “.” indicates semi-conserved residues.FIG. 9B is an image depicting the structural model of E23A. The PDBcoordinates generated by CPHModels-3.0 were uploaded onto FirstGlance inJmol to visualize the structural model. The N-terminal alpha helix andbeta strands are shown as ribbons, with arrowheads pointing towards thecarboxy termini. Random coil is shown as smoothed backbone traces. Theputative disulphide bond is shown as a thin cylinder.

DETAILED DESCRIPTION

The present invention provides peptide compounds that regulate theclassical and lectin pathways of the complement system, specifically bybinding and/or inactivating C1 and MBL and thus regulating the classicaland lectin pathway activation at its earliest point without affectingthe alternative pathway. These peptide compounds are of therapeuticvalue for the treatment of diseases and conditions mediated bydysregulated activation of the classical and lectin pathways.

The invention is based on the identification of an isolated, purifiedpeptide of 30 amino acids derived from human astrovirus coat protein,termed CP1, and having a sequence (SEQ ID NO:1) that is able to regulatethe classical and lectin pathway activation by binding to C1q and MBL.In other embodiments, the peptide compounds regulate the classicalpathway activation but not the lectin pathway activation.

Modifications of the amino acid structure of CP1 has led to thediscovery of additional peptide compounds that are able to regulate C1qactivity.

The term “peptide compound(s),” as used herein, refers to amino acidsequences, which may be naturally occurring, or peptide mimetics,peptide analogs and/or synthetic derivatives of about 30 amino acidsbased on SEQ ID NO:1. In addition, the peptide compound may be less thanabout 30 amino acid residues, such as between about 20 and about 30amino acid residues and such as peptide compounds between about 10 toabout 20 amino acid residues. Peptide residues of, for example, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, and 30 amino acids are equally likely to be peptidecompounds within the context of the present invention.

The disclosed peptide compounds are generally constrained (that is, havesome element of structure as, for example, the presence of amino acidsthat initiate a β turn or β pleated sheet, or, for example, are cyclizedby the presence of disulfide bonded Cys residues) or unconstrained (thatis, linear) amino acid sequences of about 30 amino acid residues, orless than about 30 amino acid residues.

Substitutes for an amino acid within the peptide sequence may beselected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine. Amino acids containing aromatic ringstructures include phenylalanine, tryptophan, and tyrosine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine, and glutamine. The positively charged (basic)amino acids include arginine and lysine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid of a similar polarity, which acts as a functionalequivalent, resulting in a silent alteration.

A conservative change generally leads to less change in the structureand function of the resulting protein. A non-conservative change is morelikely to alter the structure, activity, or function of the resultingprotein. For example, the peptide of the present disclosure comprisesone or more of the following conservative amino acid substitutions:replacement of an aliphatic amino acid, such as alanine, valine,leucine, and isoleucine, with another aliphatic amino acid; replacementof a serine with a threonine; replacement of a threonine with a serine;replacement of an acidic residue, such as aspartic acid and glutamicacid, with another acidic residue; replacement of a residue bearing anamide group, such as asparagine and glutamine, with another residuebearing an amide group; exchange of a basic residue, such as lysine andarginine, with another basic residue; and replacement of an aromaticresidue, such as phenylalanine and tyrosine, with another aromaticresidue.

Particularly preferred amino acid substitutions include:

a) Ala for Glu or vice versa, such that a negative charge may bereduced;

b) Lys for Arg or vice versa, such that a positive charge may bemaintained;

c) Ala for Arg or vice versa, such that a positive charge may bereduced;

d) Glu for Asp or vice versa, such that a negative charge may bemaintained;

e) Ser for Thr or vice versa, such that a free —OH can be maintained;

f) Gln for Asn or vice versa, such that a free NH2 can be maintained;

g) Ile for Leu or for Val or vice versa, as roughly equivalenthydrophobic amino acids;

h) Phe for Tyr or vice versa, as roughly equivalent aromatic aminoacids; and

i) Ala for Cys or vice versa, such that disulphide bonding is affected.

In one embodiment, the invention discloses an isolated, purified peptidederived from human astrovirus coat protein, the peptide comprising theamino acid sequence of SEQ ID NO: 1.

In another embodiment, the invention discloses an isolated, purified,synthetic peptide comprising the amino acid sequence of SEQ ID NO: 1,with one or more amino acid substitutions, modifications, insertions, ordeletions, wherein the peptide regulates complement activation.

In another embodiment, the invention discloses an isolated, purified,synthetic peptide comprising the amino acid sequence of SEQ ID NO: 1,with one or more conservative amino acid substitutions, wherein thepeptide regulates complement activation.

The peptide compounds may have internal peptide deletions andsubstitutions as well as deletions and substitutions at the N-terminusand C-terminus based on SEQ ID NO:1. In some embodiments, the peptidehas about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more amino acidsubstitutions, modifications, insertions, or deletions.

In some embodiments, the peptide sequence has at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity to SEQID NO:1.

Astrovirus Coat Protein Peptides and Derivatives

Two 30-residue peptides that encompassed the region of homology betweenHNP-1 and the WT CP, CP1 and CP2, were synthesized (FIG. 2A). CP1demonstrated consistent regulation of classical pathway activation,while CP2 did not regulate classical pathway activation. CP1 retainslimited homology with HNP-1 at its N and C termini (four residues each);however, no homology exists internally to these flanking residues, asshown in FIG. 2A. While not bound by any theory, WT CP may regulate C1and MBL activity by binding the collagen-like regions of both moleculesand dissociating their associated serine proteases, C1s-C1r-C1r-C1s andMASP2, from C1q and MBL, respectively (Hair et al., 2010. Molec.Immunol. 47, 792-798).

The peptide compounds in this disclosure were synthesized throughpeptide deletions and substitutions of CP1, the 30 amino acid peptide ofHAstV CP described above. Additional peptide compounds have beensynthesized based on modifications of CP1, as shown in TABLE 1 below.

TABLE 1 Peptide Peptide sequence (N → C) CP2NPVLVKDATGSTQFGPVQALGAQYSMWKLK (SEQ ID NO: 2) CP1PAICQRATATLGTVGSNTSGTTEIEACILL (SEQ ID NO: 1) C04A PAI AQRATATLGTVGSNTSGTTEIEACILL (SEQ ID NO: 3) C27APAICQRATATLGTVGSNTSGTTEIEA A ILL (SEQ ID NO: 4) E23APAICQRATATLGTVGSNTSGTT A IEACILL (SEQ ID NO: 5) E25APAICQRATATLGTVGSNTSGTTEI A ACILL (SEQ ID NO: 6) Δ8-22PAICQRA---------------EIEACILL (SEQ ID NO: 7) Δ8-22 oxidizedPAICQRA---------------EIEACILL (SEQ ID NO: 7) Δ8-22 AbuPAIaQRA---------------EIEAaILL (SEQ ID NO: 8) Polar AssortantIALILEPICCQERAA (SEQ ID NO: 9)

CP1 is an isolated, purified peptide derived from human astrovirus coatprotein, the peptide comprising an amino acid sequence of SEQ ID NO: 1.

CP2 is an isolated, purified peptide derived from human astrovirus coatprotein, the peptide comprising an amino acid sequence of SEQ ID NO: 2.

Using CP1 as the parent peptide, four alanine substitutions (C04A, C27A,E23A, E25A) were made (alanine-substituted residues are in bold andunderlined in TABLE 1). Internal deletions of residue 8 to residue 22were made for the Δ8-22 peptide (internal deletions are shown as dashesin TABLE 1). CP2, CP1, and CP1 derivatives were coated on a plate. Afterblocking, increasing amounts of C1q was added for 1 hour at roomtemperature, followed by detection of C1q with antisera to C1q. Bindingof C1q by the various peptide derivatives is shown in FIG. 3. Datarepresents triplicate readings for each peptide compound. CP1 bound C1qin a dose-dependent manner (FIG. 3A). While CP2 bound less C1q than CP1,it bound C1q at similar levels to that of HNP-1 (FIG. 3A).

As shown in TABLE 1, CP1 contains two cysteine residues at positions 4and 27, which were individually substituted with alanine in C04A andC27A. The cysteine residues of CP1 were targeted to determine ifdisulphide bonding in CP1 is required for classical pathway activation.As is known in the art, the peptide regulators of C3 (Compstatin) andC1q (peptide 2J), both of which contain two cysteine residues in theiramino acid sequences, require cyclicalization through disulphide bondingfor activity (Sahu et al., 1996. J. Immunol. 157, 884-891; Roos et al.,2001. J. Immunol. 167, 7052-7059). The cysteine substitutions had nosignificant effects on C1q binding (FIG. 3B) or C4 activation (FIG. 5).However, both cysteine substitutions demonstrated loss of complementregulatory activity in the hemolytic assays (FIG. 6B). This suggeststhat although cyclicalization via disulphide bonding of the cysteineresidues is not critical for C1q binding, cyclicalization appears tohave a variable effect on their ability to inhibit activation of thecomplement system. Thus, while not bound by any theory, disulphidebonding of the cysteine residues may be important for proper peptideconformation and stability, as suggested by the structural model of E23A(FIG. 9B).

As shown in TABLE 1, glutamic acid residues at positions 23 and 25 werealso substituted with alanine. Substitution of the glutamic acidresidues was performed because these negatively charged amino acids mayplay a role in CP interaction with non-hydroxylated lysine residues onthe C1q molecule. E23A and E25A peptides demonstrated efficient bindingto C1q (FIG. 3C) and similar or greater regulatory activity than CP1 inall functional assays (FIGS. 5 and 6). In particular, E23A showedsuperior regulation of classical pathway activation in comparison to allthe other peptide derivatives tested. It appears that substitution ofone of the negatively charged glutamic acid by the neutral alanineresidue enhances the peptide's regulatory activity.

As shown in TABLE 1, the Δ8-22 peptide was a deletion of residues 8-22from E23A. This peptide was active in all functional assays tested andbound C1q (FIG. 3D). This Δ8-22 peptide retains the two cysteine and twoglutamic acid residues and is half the size of CP1 (15 residues versus30 residues).

The Δ8-22 peptide (SEQ ID No. 7) was oxidized during synthesis to form adisulphide bond between the two cysteine residues (Δ8-22 oxidized). Thispeptide was active in all functional assays tested. The two cysteineresidues were replaced in the Δ8-22 peptide with a cysteine derivativethat does not form a disulphide bond, such that the peptide staysreduced. (Δ8-22 peptide Abu; SEQ ID No. 8). This peptide was active inall functional assays tested. With the peptide Polar Assortant (SEQ IDNo. 9), the 15 amino acid residues from Δ8-22 peptide were scrambled.This peptide was also active in all functional assays tested.

Rational Peptide Deletions, Substitutions, and Modifications

A series of peptide deletions, substitutions, and modifications of CP1are disclosed, as shown in TABLE 2 below. The subject matter disclosesan isolated, purified, synthetic peptide comprising any one of the aminoacid sequences of SEQ ID NOs: 1-35, as shown in TABLES 1 and 2.

TABLE 2 Peptide

(SEQ ID NO: 1) PAICQRATATLGT---NTSGTTEIEACILL (SEQ ID NO: 10)PAICQRATATL-------SGTTEIEACILL (SEQ ID NO: 11)PAICQRATA-----------TTEIEACILL (SEQ ID NO: 12) Δ8-22PAICQRA---------------EIEACILL (SEQ ID NO: 7) N-terminal deletions--AICQRATATLGTVGSNTSGTTEIEACILL (SEQ ID NO: 13)--ICQRATATLGTVGSNTSGTTEIEACILL (SEQ ID NO: 14)---CQRATATLGTVGSNTSGTTEIEACILL (SEQ ID NO: 15) C-terminal deletionsPAICQRATATLGTVGSNTSGTTEIEACIL- (SEQ ID NO: 16)PAICQRATATLGTVGSNTSGTTEIEACI-- (SEQ ID NO: 17)PAICQRATATLGTVGSNTSGTTEIEAC--- (SEQ ID NO: 18)N- and C-terminal deletions CP1, aa 8-22 TATLGTVGSNTSGTT (SEQ ID NO: 19)CP2, aa 9-23 TGSTQFGPVQALGAQ (SEQ ID NO: 20) Alanine scan including thefollowing specific substitutions C04A PAI A QRATATLGTVGSNTSGTTEIEACILL(SEQ ID NO: 3) C27A PAICQRATATLGTVGSNTSGTTEIEA A ILL (SEQ ID NO: 4)C04,27A PAIAQRATATLGTVGSNTSGTTEIEA A ILL (SEQ ID NO: 21) E23APAICQRATATLGTVGSNTSGTT A IEACILL (SEQ ID NO: 5) E25APAICQRATATLGTVGSNTSGTTEI A ACILL (SEQ ID NO: 6) E23,25APAICQRATATLGTVGSNTSGTT A I A ACILL (SEQ ID NO: 22) E23A, Δ8-22 AAICQRA---------------EIEACILL (SEQ ID NO: 23) E23A, Δ8-22 PA ACQRA---------------EIEACILL (SEQ ID NO: 24) E23A, Δ8-22 PAI AQRA---------------EIEACILL (SEQ ID NO: 25) E23A, Δ8-22 PAIC ARA---------------EIEACILL (SEQ ID NO: 26) E23A, Δ8-22 PAICQ AA---------------EIEACILL (SEQ ID NO: 27) E23A, Δ8-22PAICQRA--------------- A IEACILL (SEQ ID NO: 28) E23A, Δ8-22PAICQRA---------------E A EACILL (SEQ ID NO: 29) E23A, Δ8-22PAICQRA---------------EI A ACILL (SEQ ID NO: 30) E23A, Δ8-22PAICQRA---------------EIEA A ILL (SEQ ID NO: 31) E23A, Δ8-22PAICQRA---------------EIEAC A LL (SEQ ID NO: 32) E23A, Δ8-22PAICQRA---------------EIEACI A L (SEQ ID NO: 33) E23A, Δ8-22PAICQRA---------------EIEACIL A (SEQ ID NO: 34) N-terminal acetylation

(SEQ ID NO: 35)Internal Deletions

As described above, CP1 is 30 amino acid residues in length and alignswith the first ten residues of HNP-1, as shown in FIG. 2A. Alignment ofthese two molecules was based upon the cysteine residues at the N and Cterminus that are required for cyclicalization of CP1. This led to an18-residue internal region of CP1 that shares no sequence homology withHNP-1. Increasingly large internal deletions of CP1 are synthesized andevaluated for C1q and MBL binding (TABLE 2, Internal deletions).

N- and C-Terminal Deletions

As shown in TABLE 2 (N- and C-terminal deletions), the N and C terminalamino acids are progressively deleted individually up to each cysteineresidue of CP1. Additionally, both N- and C-terminal deletions from CP1and CP2 are made to create 15 amino acid peptides. These modifiedpeptide compounds are synthesized and evaluated for C1q and MBL bindingto determine if these flanking residues are required for C1q and MBLbinding activity. These deletions assist in determining the minimal sizeof the peptide compound required for regulating complement activation.

Alanine Scan

Alanine scanning is done to identify specific amino acid residues thatare responsible for a peptide's activity. With alanine scanning, alanineis used to substitute each residue sequentially. The substitution of anessential amino acid results in a change in peptide activity, with thedegree of activity taken as a relative measure of the importance of theamino acid being substituted.

The subject matter discloses peptide compounds substituted with alanineat certain positions. The subject matter discloses an isolated,purified, synthetic peptide comprising the sequence of SEQ ID NO:1,wherein one or more of the amino acids are substituted with alanine,wherein the peptide regulates complement activation. In one or moreembodiments, the amino acids substituted with alanine are at positions4, 23, 25, or 27.

Two glutamic acid residue positions are substituted both individuallyand together with alanine (TABLE 2, in bold and underlined). While notbound by any theory, the wild-type CP molecule may interact withreactive lysine residues on C1q and MBL that are required for bindingthe serine proteases C1s-C1r-C1r-C1s and MASP2, respectively. Given thenegative charge associated with glutamic acid, these residues mayinteract directly with the positively charged lysine residues on C1q andMBL to facilitate CP binding. Two cysteine residue positions, bothindividually and together, are substituted with alanine (TABLE 2, inbold and underlined).

N-Terminal Acetylation

The subject matter discloses peptide compounds with acetylation of the Nterminus. Acetylation of CP1 increases potency by reducing the charge atthe N terminus of the peptide (i.e., electrostatic effects) (Ricklin andLambris, 2008. Nat. Biotech. 25, 1265-1275). This modification may aidin improving the in vivo stability of the peptide with respect toexopeptidases, as was shown with Compstation (Ricklin and Lambris, 2008.Nat. Biotech. 25, 1265-1275). The peptide compound synthesized fromN-terminal acetylation includes any of the peptides described above,wherein the peptide is modified through acetylation of the N-terminalresidue.

The Complement System and Diseases Associated with its Dysregulation

While complement is a vital host defense against pathogenic organismssuch as bacteria and some enveloped viruses, its unchecked activationcan cause devastating host cell damage. Host tissue damage mediated bycomplement has been implicated in a wide variety of diseases, includingautoimmune pathologies such as: rheumatoid arthritis, systemic lupuserythematosus, multiple sclerosis, myasthenia gravis, autoimmunehemolytic anemia, membranoproliferative glomerulonephritis, and serumsickness. It has also been identified as contributing to thepathogenesis of the following diseases: Adult Respiratory DistressSyndrome (ARDS), ischemia-reperfusion injuries (including stroke andmyocardial infarction), allo- and xeno-transplantation complications(including hyperacute rejection and graft versus host disease (GVHD)),Alzheimer's disease, burn injuries, hemodialysis damage, cardiopulmonarybypass damage, and paroxysmal nocturnal hemoglobinuria (PNH).

Hereditary angioedema (HAE) is a very rare genetic disorder caused byreduced levels of or non-functional C1-inhibitor; symptoms of HAEinclude acute edema. C1-inhibitor naturally regulates C1 activation, andtreatment of acute edema requires substantial infusion of C1-inhibitoror plasma transfusion. Because astrovirus CP functionally blocks C1activation, using the disclosed peptide compounds to treat HAE fulfillsa therapeutic need because C1-inhibitor has to be purified from humansera from multiple subjects and, therefore, could be contaminated withhuman blood-borne pathogens. Therapeutic administration of the disclosedpeptide compounds regulates C1 either in adjunct therapy withC1-inhibitor or as a stand-alone therapeutic treatment.

The disclosed peptide compounds can selectively regulate C1q and MBLactivation without affecting alternative pathway activity and are, thus,ideal for preventing and treating diseases mediated by the dysregulatedactivation of the classical and lectin pathways. The alternative pathwayis essential for immune surveillance against invading pathogens, andhumans with alternative pathway defects suffer severe bacterialinfections. By binding and inactivating C1q and MBL, the peptidecompounds can efficiently regulate classical and lectin pathwayactivation while leaving the alternative pathway intact.

The term “regulate,” as used herein, refers to i) controlling, reducing,inhibiting or regulating the biological function of an enzyme, protein,peptide, factor, byproduct, or derivative thereof, either individuallyor in complexes; ii) reducing the quantity of a biological protein,peptide, or derivative thereof, either in vivo or in vitro; or iii)interrupting a biological chain of events, cascade, or pathway known tocomprise a related series of biological or chemical reactions. The term“regulate” may thus be used, for example, to describe reducing thequantity of a single component of the complement cascade compared to acontrol sample, reducing the rate or total amount of formation of acomponent or complex of components, or reducing the overall activity ofa complex process or series of biological reactions, leading to suchoutcomes as cell lysis, formation of convertase enzymes, formation ofcomplement-derived membrane attack complexes, inflammation, orinflammatory disease. In an in vitro assay, the term “regulate” mayrefer to the measurable change or reduction of some biological orchemical event, but the person of ordinary skill in the art willappreciate that the measurable change or reduction need not be total tobe “regulatory.”

Pharmaceutical Formulation and Administration

The present disclosure provides pharmaceutical compositions capable ofregulating the complement system, comprising at least one peptidecompound, as discussed above, and at least one pharmaceuticallyacceptable carrier, diluent, or excipient. Pharmaceutically acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed. They can be solid, semi-solid, orliquid. The pharmaceutical compositions of the present invention can bein the form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, or syrups.

Some examples of pharmaceutically acceptable carriers, diluents, orexcipients include: lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, sterile water, syrup, and methyl cellulose. Thepharmaceutical compositions of the present invention can be formulatedusing procedures known in the art to provide quick, normal, or sustainedor delayed release of the active ingredient.

The disclosure relates to a method of regulating the complement systemin a subject comprising administering to a subject the compositionsdescribed above. The pharmaceutical compositions of the presentinvention are prepared by mixing the peptide compound having theappropriate degree of purity with pharmaceutically acceptable carriers,diluents, or excipients. Examples of formulations and methods forpreparing such formulations are well known in the art. Thepharmaceutical compositions of the present invention are useful as aprophylactic and therapeutic agent for various disorders and diseases,as set forth above. In one embodiment, the composition comprises atherapeutically effective amount of the peptide compound. In anotherembodiment, the composition comprises at least one other activeingredient effective in treating at least one disease associated withcomplement-mediated tissue damage. The term “therapeutically effectiveamount,” as used herein, refers to the total amount of each activecomponent that is sufficient to show a meaningful benefit to thesubject.

The term “subject,” as used herein, means any subject for whomdiagnosis, prognosis, or therapy is desired. For example, a subject canbe a mammal, e.g., a human or non-human primate (such as an ape, monkey,orangutan, or chimpanzee), a dog, cat, guinea pig, rabbit, rat, mouse,horse, cattle, or cow.

As used herein, “treat,” “treating,” or “treatment” refers toadministering a therapy in an amount, manner (e.g., schedule ofadministration), and/or mode (e.g., route of administration), effectiveto improve a disorder (e.g., a disorder described herein) or a symptomthereof, or to prevent or slow the progression of a disorder (e.g., adisorder described herein) or a symptom thereof. This can be evidencedby, e.g., an improvement in a parameter associated with a disorder or asymptom thereof, e.g., to a statistically significant degree or to adegree detectable to one skilled in the art. An effective amount,manner, or mode can vary depending on the subject and may be tailored tothe subject. By preventing or slowing progression of a disorder or asymptom thereof, a treatment can prevent or slow deterioration resultingfrom a disorder or a symptom thereof in an affected or diagnosedsubject.

The therapeutically effective amount of the peptide compound variesdepending on several factors, such as the condition being treated, theseverity of the condition, the time of administration, the route ofadministration, the rate of excretion of the compound employed, theduration of treatment, the co-therapy involved, and the age, gender,weight, and condition of the subject, etc. One of ordinary skill in theart can determine the therapeutically effective amount. Accordingly, oneof ordinary skill in the art may need to titer the dosage and modify theroute of administration to obtain the maximal therapeutic effect.

The effective daily dose generally is within the range of from about0.001 to about 100 milligrams per kilogram (mg/kg) of body weight,preferably about 0.01 to about 50 mg/kg, more preferably about 0.1 toabout 20 mg/kg. This dose can be achieved through a 1-6 time(s) dailydosing regimen. Alternatively, optimal treatment can be achieved througha sustained release formulation with a less frequent dosing regimen.

Pharmaceutical formulations may be adapted for administration by anyappropriate route, for example, by the oral, nasal, topical (includingbuccal, sublingual, or transdermal), or parenteral (includingsubcutaneous, intracutaneous, intramuscular, intraarticular,intraperitoneal, intrasynovial, intrasternal, intrathecal,intralesional, intravenous, or intradermal injections or infusions)route. For human administration, the formulations preferably meetsterility, pyrogenicity, general safety, and purity standards, asrequired by the offices of the Food and Drug Administration (FDA).

Combination Therapies

A further embodiment of the invention provides a method of preventing ortreating a disease associated with complement-mediated tissue damage,comprising administering to a subject the pharmaceutical compositions ofthe present invention. While the pharmaceutical compositions of thepresent invention can be administered as the sole active pharmaceuticalagent, they can also be used in combination with one or more therapeuticor prophylactic agent(s) that is(are) effective for preventing ortreating the disease. In this aspect, the method of the presentinvention comprises administrating the pharmaceutical composition of thepresent invention before, concurrently, and/or after one or moreadditional therapeutic or prophylactic agents effective in treating atleast one disease associated with complement-mediated tissue damage.

For example, the pharmaceutical compositions of the present inventioncan be used to treat rheumatoid arthritis, either alone or incombination with a non-steroidal anti-inflammatory agent (NSAID), acorticosteroid, or a disease modifying anti-rheumatic drug (DMARD).

Examples of NSAIDs include: salicylates (such as aspirin, amoxiprin,benorilate, choline magnesium salicylate, diflunisal, faislamine, methylsalicylate, magnesium salicylate, and salicyl salicylate (salsalate)),arylalkanoic acids (such as diclofenac, aceclofenac, acemetacin,bromfenac, etodolac, indometacin, ketorolac, nabumetone, sulindac, andtolmeti), 2-arylpropionic acids (such as ibuprofen, carprofen, fenbufen,fenoprofen, flurbiprofen, ketoprofen, loxoprofen, naproxen, tiaprofenicacid, and suprofen), N-arylanthranilic acids (such as mefenamic acid andmeclofenamic acid), pyrazolidine derivatives (such as phenylbutazone,azapropazone, metamizole, oxyphenbutazone, and sulfinprazone), oxicams(such as piroxicam, lornoxicam, meloxicam, and tenoxicam), COX-2inhibitors (such as etoricoxib, lumiracoxib, and parecoxib),sulphonanilides such as nimesulide, and others such as licofelone andomega-3 fatty acids.

Examples of corticosteroid include: triamcinolone (Aristocort®),cortisone (Cortone® Acetate Tablets), dexamethasone (Decadron® Elixir),prednisone (Deltasone®), and methylprednisolone (Medrol®).

Examples of DMARD include: methotrexate (Rheumatrex®), leflunomide(Arava®), etanercept (Enbrel®), infliximab (Remicade®), adalimumab(Humira®), anakinra (Kineret®), sulfasalazine (Azulfidine EN-Tabs®),antimalarials, gold salts, d-penicillamine, cyclosporin A,cyclophosphamide and azathioprine.

Soliris™ (eculizumab) is a humanized anti-C5 monoclonal antibody. It hasbeen approved by the FDA for the treatment of the rare form of hemolyticanemia, paroxysmal nocturnal hemoglobinuria. In one embodiment, thepharmaceutical compositions of the present invention can be used incombination with Soliris™ in treating paroxysmal nocturnalhemoglobinuria, heart disease, pulmonary diseases, autoimmune diseases,asthma, as well as the ancillary care of transplants.

The pharmaceutical compositions of the present invention can beadministered with the additional agent(s) in combination therapy, eitherjointly or separately, or by combining the pharmaceutical compositionsand the additional agent(s) into one composition. The dosage isadministered and adjusted to achieve maximal management of theconditions. For example, both the pharmaceutical compositions and theadditional agent(s) are usually present at dosage levels of betweenabout 10% and about 150%, more preferably, between about 10% and about80%, of the dosage normally administered in a mono-therapy regimen.

EXAMPLES

The invention is further illustrated by the following examples, providedfor illustrative purposes only. They are not to be construed as limitingthe scope or content of the invention in any way.

Materials and Methods

Example 1—Preparation of HAstV-1 CP, Peptides, Heat-Aggregated IgG,Sera, Erythrocytes, and Complement Buffers

Wild-type HAstV-1 CP was expressed from a recombinant baculovirus inSpodoptera frugiperda cells (line IPLB-Sf21) and purified as previouslydescribed (Bonaparte et al., 2008. J. Virol. 82, 817-827). HNP-1, CP1,and CP2 peptides were obtained from Biomatik, whereas C04A, C27A, E23A,E25A and d8-22 were purchased from GenScript. Before shipment, thepeptide compounds were analyzed by HPLC and ESI-mass spectrometry. Uponreceipt, peptides were dissolved in dimethylsulfoxide (DMSO) at aconcentration of 10 mM and stored at −80° C. Heat-aggregated human IgGwas prepared using methods known in the art (Bonaparte et al., 2008. J.Virol. 82, 817-827). Pooled normal human serum (NHS) was derived fromthe blood of healthy human volunteers according to an InstitutionalReview Board approved protocol (IRB 02-06-EX-0216, Eastern VirginiaMedical School) and was pooled, aliquoted, and frozen at −80° C. usingmethods known in the art (Cunnion et al., 2001. Infect. Immun. 69,6796-6803). Antibody-sensitized sheep erythrocytes were generated usingmethods known in the art (Bonaparte et al., 2008. J. Virol. 82,817-827). Standard complement buffers were used: GVBS⁺⁺(Veronal-buffered saline, 0.1% gelatin, 0.15 mM CaCl₂, and 1.0 mM MgCl₂)and GVBS⁻⁻ (Veronal-buffered saline, 0.1% gelatin, 0.01 M EDTA).

Example 2—C1q ELISA Protocols for WT CP and Peptide Compounds

To analyze whether CP competes with HNP-1 for C1q binding, HNP-1peptides (2.5 μM) were coated onto 96-well Maxisorp plates (Nunc) incoating buffer (100 mM Na₂CO₃, NaHCO₃, [pH 9.6]) and plates wereincubated at room temperature overnight. Plates were washed with PBS/Tand blocked with 3% BSA/PBS, 0.05% Tween-20 (PBS/T) for 2 hours at roomtemperature. Next, a constant amount of purified C1q (10 μg/ml;Complement Technologies, Inc.) was added to each well, while decreasingamounts of CP, starting at 100 μg/mL, were added simultaneously andincubated for 1 hour at room temperature. BSA was substituted for CP asa negative control for competition. After washing, the primary antibody,goat anti-C1q polyclonal antibody (Complement Technologies, Inc.), wasdiluted 1:2,000 in 3% BSA/PBS/T and added to the plate for 1 hour atroom temperature. The plate was washed and the secondary antibody,donkey anti-goat HRP (Santa Cruz Biotechnology, Inc.), was diluted1:2,500 in 3% BSA/PBS/T and incubated for 1 hour at room temperature.The plates were washed with PBS/T and developed with tetramethylbenzindine (Sigma) for 1 min. The reactions were then stopped with 0.1ml 1N H₂SO₄ and absorbance was read in a Synergy HT plate reader(Bio-Tek Instruments) at a wavelength of 450 nm. To assess competitivebinding of CP peptides CP1 and CP2 for C1q binding, the assay wascarried out in an identical manner to that described above, except CP1and CP2 were coated onto plates (2.5 μM) and BSA was used in parallelwith CP as a negative control for competition.

To determine the binding of CP peptide derivatives to C1q, peptides at2.5 μM were coated on the plate and incubated overnight at roomtemperature. After washing and blocking, decreasing amounts of C1q,starting at 100 μg/mL, were added to the wells and incubated for 1 hourat room temperature. C1q was detected and the plates developed asdescribed above.

Example 3—C1s Immunoblot

One μl of partially purified human C1 (0.2 mg/ml, ComplementTechnologies, Inc.) was incubated at 37° C. for 90 minutes, either aloneor with heat-aggregated human immunoglobulin G (5 μl of a 1:250 dilutionof 50 μg/ml starting solution), or with increasing amounts of theindicated peptides (250 μM stock), and brought up to a total volume of11 μl in PBS. After the incubation, an equal volume of loading bufferwas added to all samples, which were subsequently boiled andelectrophoresed through an 8% SDS-PAGE for 60 minutes at 140 volts. Thegel was then transferred to nitrocellulose and blocked with non-fatdried milk (NFDM) in PBS. The blot was probed with a goat polyclonalantibody to C1s (Quidel) at a 1:2,000 dilution, washed in PBS/0.1%Tween-20, followed by HRP-conjugated donkey anti-goat IRDye 680 antibody(Li-cor Biosciences) at a 1:10,000 dilution and washed with PBS/0.1%Tween. The blot was then imaged on an Odyssey imager using version 3.0software (Li-cor Biosciences), and activation of C1s was determined fromthe amounts of the C1s heavy and light chains characteristic ofactivated C1s relative to the proenzyme species.

Example 4—C4 Activation Assay

The C4 activation assay was adapted from Mallik et al., 2005. J. Med.Chem. 48, 274-286. Wells of Immulon-2, 96 well plates were coated with50 μl of 1.0 mg/ml ovalbumin (Fisher) in coating buffer and incubatedovernight at 4° C. The plates were washed with PBS/T and blocked with 3%BSA/PBS for 2 hours at room temperature. The plates were washed againand then incubated with a rabbit anti-ovalbumin antibody (Millipore)diluted in 3% BSA/PBS at 1:2,000 for 1 hour at room temperature. Duringthis incubation, the peptides were diluted to 0.5 mM in 10% NHS/GVBS⁺⁺and incubated for 15 minutes at 37° C. The plates were then washed, andthe pre-incubated samples were added to the plates at a 1:4 dilution inGVBS⁺⁺ and incubated for 30 minutes at room temperature. Afterwards, theplates were washed and goat anti-C4 antibody (Complement Technologies,Inc.) was added at a dilution of 1:2,000 in 3% BSA/PBS for 1 hour,followed by another wash and a donkey anti-goat IgG-HRP antibody (SantaCruz Biotechnology, Inc.) diluted to 1:2,000 in 3% BSA/PBS for 1 hour.The plates were then developed and absorbance values determined asdescribed above.

Example 5—Hemolytic Assay

Peptides were diluted to 1.4 mM or 0.77 mM in undiluted NHS or factorB-depleted human sera (Complement Technologies, Inc.) and incubated for1 hour at 37° C. These peptides were then diluted with GVBS⁺⁺ to equal2.5% NHS, of which 0.25 ml was combined with 0.4 ml of GVBS⁺⁺ and 0.1 mlof sensitized sheep red blood cells (RBCs) and again incubated for 1hour at 37° C. The procedure was stopped by the addition of 4.0 ml ofGVBS⁻⁻, centrifuged for 5 minutes at 1,620×g, and the absorbance of thesupernatants was read at 412 nm in a spectrophotometer. The percentlysis of each sample was standardized to that of the NHS only control.

Example 6—Hemolytic Assay Titration of Polar Assortant Peptide in FactorB-Depleted Serum

Polar Assortant peptide was serially diluted as indicated in FIG. 7 inundiluted factor B-depleted human sera (Complement Technologies, Inc.)and incubated for 1 hour at 37° C. Factor B-depleted serum alone, 0.77mM of Δ8-22 and DMSO were included as controls. These peptides were thendiluted with GVBS⁺⁺ to equal 2.5% NHS, of which 0.25 ml was combinedwith 0.4 ml of GVBS⁺⁺ and 0.1 ml of sensitized sheep red blood cells(RBCs) and again incubated for 1 hour at 37° C. The procedure wasstopped by the addition of 4.0 ml of GVBS⁻⁻, centrifuged for 5 minutesat 1,620×g, and the absorbance of the supernatants was read at 412 nm ina spectrophotometer. The percent lysis of each sample was standardizedto that of the NHS only control.

Example 7—Statistical Analysis

For replicate experiments, means and standard errors of the mean (SEMs)were calculated using techniques known in the art (Microsoft Excel XP).

Example 8—Mass Spectrometric Analysis of Peptide Oligomerization

Synthetic peptides were purified by C18 ZipTips™ (Millipore) before massspectrometry analysis, as follows: 10 μl of 70% acetonitrile (ACN)/0.1%triflouroacetic acid (TFA) was pipetted two times through the ZipTip towet the resin, followed by two 10 μl washes of 0.1% TFA to equilibratethe resin. The acidified peptide sample was aspirated up and down fivetimes through the ZipTip to bind peptides to the resin. Contaminantswere washed by pipetting 0.1% TFA three times through the ZipTip beforeeluting the bound peptides into a fresh tube using 70% ACN/0.1% TFA. Thepeptides were dried in a speed vac, re-suspended in 10 μl 0.1% TFAbefore mixing with matrix (α-cyano-4 hydroxycinnamic acid or sinapinicacid) at a ratio of 1:4 before analysis. Mass spectrometry was performedusing a Bruker Daltonics Ultraflex II™ MALDI-TOF-TOF and the dataacquired in both the reflectron and linear positive modes.

Example 9—Homology Modeling of E23A Peptide

The amino acid sequence of E23A (SEQ ID NO: 5) was uploaded onto theCPHmodels 3.0 server Lund et al., 2002. Abstract at the CASP5 conferenceA102). The program aligned E23A with the Vigna radiata plant defensin 1(VrD1), which provided the template and Protein Data Bank (PDB)coordinates for generating the structural model. The PDB coordinates forthe E23A were subsequently uploaded onto FirstGlance in JMol, version1.45 to visualize the structure.

Results Example 10—CP Competes with HNP-1 Peptide for C1q Binding

Earlier studies have demonstrated that CP expressed as a recombinantbaculovirus-expressed protein and purified from insect cell lysates canefficiently bind C1q and MBL with resultant inhibition of the classicaland lectin complement pathways (Bonaparte et al., 2008, Hair et al.,2010). Previously, it was shown that the peptide human neutrophildefensin-1 (HNP-1) can bind C1q and MBL, regulating activation of theclassical and lectin pathways of complement, respectively (van den Berget al., 1998. Blood. 92, 3898-3903; Groeneveld et al., 2007. Molec.Immunol. 44, 3608-3614). Given that CP also possesses these properties,the amino acid sequences of both proteins were analyzed and a region oflimited homology found between HNP-1 and residues 79-139 of the WT CP.Then, it was analyzed whether CP could directly compete with HNP-1 forbinding to C1q using a competition ELISA approach in which HNP-1 peptidewas coated on the ELISA plate. FIG. 1 is a graph depicting CPdose-dependently competing with human neutrophil defensin 1 (HNP-1) forbinding to C1q. A fixed amount of purified C1q and increasing amounts ofCP were added simultaneously. Adherent C1q bound to HNP-1 was detectedwith polyclonal antibody to C1q. Bound C1q signal decreased withincreasing amounts of CP, indicating that CP dose-dependently competeswith HNP-1 for C1q binding (FIG. 1, shown as circles). In contrast, whenBSA was substituted for CP, no competition for binding of C1q wasdetected (FIG. 1, shown as triangles), and the same lack of competitionwas seen with aldolase and egg albumin (data not shown). In addition, nobinding was seen when BSA was coated on the plate in place of HNP-1.Although not bound by any theory, this data is consistent with HNP-1 andCP binding C1q in a comparable manner to regulate classical pathwayactivation (Hair et al., 2010. J. Virol. 82, 817-827).

Example 11—Identification of a CP Peptide with Homology to HNP-1 thatCompetes with WT CP for Binding to C1q

Because CP efficiently competed with HNP-1 for binding to C1q, it wasanalyzed whether CP and HNP-1 shared any homology at the amino acidsequence level. Alignment of the 787 amino acid CP molecule with the 30amino acid HNP-1 peptide was performed using Clustal W (Larkin et al.,2007. Bioinformatics. 23, 2947-2948). A region of homology was observedbetween HNP-1 and residues 79-139 of the CP molecule (FIG. 2A). Toascertain whether this CP sequence retained the complement regulationfunctions of WT CP, two 30 residue peptides were synthesized whichencoded residues 79-108 (CP1) and 109-138 (CP2) of the CP molecule (FIG.2A). CP1 aligned with the first 10 residues of HNP-1, whereas CP2aligned with the last 20 residues. To ascertain whether peptides CP1 andCP2 could directly bind C1q and compete with WT CP for binding to C1q, acompetition ELISA was performed in which CP1 and CP2 were coated on theELISA plate. A fixed amount of purified C1q and increasing amounts of WTCP were added simultaneously, and bound C1q was then detected withpolyclonal antibody to C1q. In the absence of CP, C1q was efficientlybound by CP1 peptide; however, the C1q signal decreased with increasingamounts of CP, indicating that CP dose-dependently competed with CP1 forC1q binding (FIG. 2B, shown as a stippled line). When BSA wassubstituted for CP under the same conditions, CP1 bound efficiently toC1q and no competition was observed (FIG. 2B, shown in triangles). Incontrast to CP1, CP2 did not compete for C1q binding (FIG. 2B, shown insquares). Thus, CP residues 79-108 were sufficient to bind C1q in asimilar manner to WT CP.

Example 12—Binding of CP Peptide Derivatives to C1q

The ability of the CP1 peptide to competitively bind C1q in a similarfashion to WT CP led to an initial analysis of the peptide residues thatare critical for these activities. Targeted amino acid substitutions anda large deletion of the parent CP1 peptide were synthesized, as shown inTABLE 1 above. C04A and C27A were designed to assess whether putativedisulphide bonding between the two cysteine residues in CP1 wererequired for C1q binding and regulating complement activation. E23A andE25A were synthesized to assess whether these negatively chargedglutamic acid residues were required for C1q binding and complementregulation. Finally, a peptide deleting internal residues 8-22 (Δ8-22)was designed to determine if this region, which does not have homologywith HNP-1, was required for C1q binding and regulating complementactivation. This peptide, Δ8-22, retained the two cysteine and twoglutamic acid residues.

To ascertain whether these peptides could bind C1q, binding assays inwhich the various peptide derivatives were coated on an ELISA plate wereperformed. Increasing amounts of purified C1q was added and bound C1qwas then detected with polyclonal antibody to C1q. CP1 dose-dependentlybound C1q (FIG. 3A), consistent with its ability to compete with WT CPfor C1q binding (FIG. 2B). CP2 bound C1q at similar levels to that ofHNP-1 (FIG. 3A), demonstrating that while this peptide does not competewith CP for C1q binding, it does retain the ability to bind C1q,possibly as a result of its homology to the C-terminal 20 amino acids ofHNP-1. The capacity of C04A and C27A to bind C1q was analyzed next, andboth peptides bound C1q similarly to CP1 (FIG. 3B). E23A and E25A werefound to bind C1q at levels between that of CP1 and CP2 (FIG. 3C), andthis trend was also observed for peptide Δ8-22 (FIG. 3D).

In summary, while all of the CP1 derivative peptides bound C1q, thedegree of binding varied depending on the amino acid substitution. Whilenot bound by any theory, neither the cysteine (C04 and C27) nor theglutamic acid (E23 and E25) residues appeared to play a critical role inC1q binding. In addition, peptide Δ8-22, which has a deletion of theinternal 15 amino acid residues of CP1, still retained C1q bindingactivity. Thus, the CP1 derivatives individually demonstrate thatbinding to C1q is not dependent on either glutamic acid residue nor oneither cysteine residue, suggesting that cyclicalization via adisulphide bond is not required.

Example 13—CP1 Peptide Regulates C1s Activation

Purified CP can regulate classical pathway activation at the level ofC1, by binding C1q and preventing the cleavage of the proenzyme C1s(Hair et al., 2010. J. Virol. 82, 817-827). To evaluate if CP1 and CP2peptides were capable of regulating C1 activation as well, partiallypurified C1 complex was incubated for 90 minutes at 37° C. withheat-aggregated IgG (a potent stimulator of classical pathwayactivation) with increasing amounts of CP1 and CP2. To evaluate C1sactivation, the cleavage of proenzyme C1s into the heavy and lightchains was detected. C1 incubated alone showed minimal spontaneousactivation of C1s, whereas C1 in the presence of heat-aggregated IgGdemonstrated robust C1s cleavage (FIG. 4A, lanes 1 and 2). Incubating C1and heat-aggregated IgG with increasing amounts of CP1 dose-dependentlysuppressed C1s cleavage (FIG. 4A, lanes 3-6) to levels observed forspontaneous C1 activation (FIG. 4A, lane 1). In contrast to CP1, CP2 didnot demonstrate significant regulation of C1s cleavage at any of theconcentrations tested (FIG. 4B, lanes 3-6). Quantification by Odysseyimaging of the regulation of C1s cleavage for CP1 and CP2 in twoindependent experiments for each peptide validated these results (FIGS.4C-4D). While CP2 demonstrated minimal binding to C1q (FIG. 3A) and didnot demonstrate the capacity to regulate C1s cleavage (FIGS. 4B and 4D),it was tested whether a combination of CP1 and CP2 would result ingreater regulation of C1s cleavage than CP1 alone. CP1 and CP2 togetherresulted in no more regulation of C1s cleavage than that observed forCP1 alone. Consistent with the ability of the peptide derivatives tobind C1q, all peptide derivatives of CP1 were also found to inhibit C1sactivation as demonstrated for CP1 (data not shown). While not bound byany theory, the ability of CP1 to regulate activation at the firstcomponent of the classical pathway, C1, suggests that this peptideregulates classical pathway activation in a comparable fashion to WT CP.

Example 14—Regulation of Complement Activity by CP Peptide in FunctionalAssays

To determine the ability of the peptide compounds to regulate complementactivation in functional assays, a C4 activation assay and a hemolyticassay were used. For the C4 activation assay, a method known in the art(Mallik et al., 2005. J. Med. Chem. 48, 274-286) was modified so thatELISA plates were coated with ovalbumin to which anti-ovalbuminantibodies were allowed to bind, mimicking an immune-complex target. Thevarious peptide compounds were then diluted to 0.5 mM in 10% NHS/GVBS⁺⁺,incubated for 15 minutes, and subsequently added to each well. Classicalpathway activation (C4) was assayed by detecting deposition ofC4-fragments using a polyclonal anti-C4 antibody. As shown in FIG. 5,NHS alone, NHS+BSA, and NHS+DMSO all demonstrated similar deposition ofC4-fragments, whereas NHS treated with WT CP regulated C4 activation.CP1 demonstrated a 35% inhibitory effect, whereas CP2 had no effect onC4 activation, consistent with the results observed for C1s activation(FIG. 4). Peptide compounds C04A, C27A, E25A, and Δ8-22 all inhibitedcomplement activation of C4 by 20-45%. E23A potently suppressed C4activation by 90%.

Peptide compound regulation of serum complement activity was assessed ina standard hemolytic complement assay. Sheep erythrocytes weresensitized with antibody and incubated with NHS, with or without peptidepre-incubation, and hemolytic complement activity was measured. Asopposed to the C4 activation assay, all three complement pathways(classical, lectin and alternative) were present and may havecontributed to the observed regulatory activity. However, initialcomplement activation was primarily driven by the antibody on theerythrocytes and, thus, the classical pathway. As demonstrated in FIG.6A, NHS either alone or in the presence of DMSO lysed erythrocytes asexpected. CP2 regulated lysis to a similar level as CP1 (66% inhibition)in contrast to the C4 activation assay, in which the CP2 peptide had noregulatory effect. C04A had minimal effect on erythrocyte lysis, whereasC27A was more inhibitory (85% inhibition). Similar to the effect seen inthe C4 activation assay, E23A inhibited erythrocyte lysis efficiently(85% inhibition) compared with E25A, which had 60% inhibition. The Δ8-22peptide compound inhibited erythrocyte lysis by 75%.

To test whether the peptides were regulating alternative pathwayactivation, the amount of serum used during the pre-incubation wasincreased, effectively lowering the concentration of the peptides testedto 0.77 mM, such that minimal inhibition occurred in NHS (FIG. 6B). Theregulatory activity of the peptides on the classical pathway alone wasthen assessed by using factor B-depleted serum in the hemolytic assay,utilizing the same amounts of peptide and serum (FIG. 6B). In contrastto the lack of regulation seen for NHS, the parental peptide CP1regulated classical complement pathway activation in factor B-depletedserum. In addition, peptide compounds E23A, E25A, and Δ8-22 regulatedclassical complement pathway activation significantly. While not boundby any theory, at higher amounts of serum, where the alternative pathwaybegins to be more efficiently activated, several peptides continued toefficiently regulate classical pathway activation, but not overallcomplement activation, suggesting the alternative pathway is mediatinghemolysis. Comparison of the regulatory activity from FIGS. 5 and 6indicate that E23A effects good regulation of complement.

The Polar Assortant peptide initially showed significant regulation ofclassical pathway activity in a hemolytic assay with NHS and factorB-depleted serum (data not shown). To further explore the regulatoryactivity of this peptide on the classical pathway, a dilution of thePolar Assortant peptide was made in factor B-depleted serum. In contrastto the lack of regulation seen for NHS alone and DMSO vehicle, the PolarAssortant peptide dose-dependently regulated classical pathwayactivation significantly beyond that of Δ8-22 (FIG. 7, compare 0.77 mMΔ8-22 versus 0.77 mM Polar Assortant).

Example 15—CP Peptides do not Oligomerize into Higher Order Structures

To further characterize the CP peptides, it was assessed whether thesecompounds could oligomerize into higher ordered structures, such asdimers, trimers, etc. To assess CP peptide oligomerization, all seven CPpeptides from TABLE 1 were analyzed by MALDI-TOF-TOF mass spectrometryin the linear and reflection modes. In both modes, all peptides werefound to be monomeric with no major peaks carrying a mass to chargeratio (m/z) greater than the theoretical mass of the peptide tested.FIGS. 8A-8B show both the linear and reflection modes, respectively.While the linear mode is lower resolution than the reflection mode, bothmodes demonstrate that E23A is monomeric with no other higher-orderpeaks evident.

Example 16—Structural Model for E23A Based on Homology with the PlantDefensin VrD1

Given the identification of E23A as a highly potent regulator ofclassical pathway activation, a structural model for the E23A peptidewas generated. The amino acid sequence of E23A was uploaded onto theCPHmodels-3.0 server. This program is a protein homology modelingresource where template recognition is based upon profile-profilealignment guided by secondary structure and exposure predictions (Lundet al., 2002. Abstract at the CASP5 conference A102). Consistent withthe homology discussed above between CP and HNP-1, CPHmodels 3.0 alignedresidues 2-29 of E23A to residues 17-44 of the 46 residue plantdefensin, Vigna radiata plant defensin 1 (VrD1) (FIG. 9A). Based uponthe nuclear magnetic resonance solution structure of VrD1 (Liu et al.,2006. Proteins. 63, 777-786), a model of the E23A was generated byCPHmodels 3.0 and displayed in FirstGlance in J Mol (FIG. 9B). FIG. 9Adepicts an 11 residue N-terminal alpha helix followed by twoanti-parallel beta strands. The alpha helix and beta strands areconnected by two, 3-5 residue disordered loops. The two cysteineresidues are shown forming a disulphide bond (depicted as a thincylinder) between the alpha helix and second beta strand, which may playa role in stabilizing the overall structure.

Rational Peptide Compound Design Example 17—Synthesis of Peptide Analogsof CP1

The peptide analogs of CP1 are commercially synthesized. These modifiedpeptides are then analyzed for interaction with C1q and MBL in bindingassays known in the art (Hair et al., 2010. Molec. Immunol. 47,792-798).). These assays are briefly described below.

Example 18—C1q Binding

CP peptide analogs are coated onto microtiter plates at variousconcentrations and analyzed for their ability to bind purified C1q(CompTech). C1q binding is detected with anti-C1q monoclonal antibody(Quidel), followed by donkey anti-mouse HRP (Santa Cruz Biotech). Theplates are then developed with tetramethyl benzindine, the reactionsterminated with H₂SO₄, and absorbance read at 450 nm. A positive controlfor C1q binding consists of CP1, whereas negative controls are BSA. Theinitial conditions for the binding of each peptide are determined, andthen serial dilutions of the peptides are performed in triplicate todetermine statistical significance and calculate half-maximal bindingvalues as previously reported (Hair et al., 2010. Molec. Immunol. 47,792-798). Half-maximal binding values are used to evaluate the relativebinding affinity of each peptide analog.

Example 19—MBL Binding

MBL binding is conducted in a similar manner to the C1q binding assaydescribed above. Purified human MBL and goat anti-MBL sera are utilized,followed by donkey anti-goat HRP for detection of MBL. Again,half-maximal binding values are calculated and compared across peptidecompounds.

The ability of CP peptide derivatives to inhibit C1 and MBL activationin functional assays is tested. The CP peptide analogs that specificallybind to C1q and MBL are assessed for their capacity to inhibit classicaland lectin pathway activation in functional assays. In addition tospecific assays for inhibition of C1 and MBL, an antibody-initiatedcomplement activation assay is utilized to determine IC50 values of thepeptide analogs in both human and rat serum. This allows for directcomparison of the relative functional activity of the peptides.

Example 20—C1 Activation Assay

The peptide compounds are analyzed for their ability to inhibit C1activation in the C1s immunoblot cleavage assay. C1 (CompTech) andheat-aggregated IgG are incubated with increasing amounts of thepeptides. C1s is detected with a goat polyclonal antibody to C1s(Quidel), followed by an infrared dye conjugated donkey anti-goatantibody (Li-Cor Biosciences) for analysis on an Odyssey infraredimaging system (Li-Cor Biosciences). C1 in the absence or presence ofheat-aggregated IgG is included on each blot as a negative and positivecontrol for C1s cleavage, respectively. To compare the extent ofinhibition of C1s cleavage by the various CP peptides, the C1s heavy andlight chains are quantified relative to C1s precursor using the Odyssey3.0 software and the percent of C1 activation is determined.CP1+heat-aggregated IgG serves as a positive control of inhibition ofC1s cleavage and can also be used to normalize values betweenexperiments, if necessary.

Example 21—MBL Activation Assay

A commercial MBL activation assay (HyCult) is used (Hair et al., 2010.Molec. Immunol. 47, 792-798) to evaluate the CP peptide analogs. Normalhuman serum (NHS) is incubated with increasing amounts of the peptidesand evaluated for lectin pathway inhibition using the commercial kit.NHS alone serves as a positive control for lectin activation, whereasheat-inactivated NHS serves as a negative control for activation. NHS+CPare used as control for demonstrating inhibition of lectin pathwayactivation.

Alternatively, a lectin activation assay known in the art (Groeneveld etal., 2007. Molec. Immunol. 44, 3608-3614) is used to evaluate thepeptide compounds.

Example 22—Antibody-Initiated Serum Complement Activation Assay

To directly compare the inhibitory activity of CP1 and its peptideanalogs, an antibody-initiated serum complement activation assay isused. This assay is a modification of protocol utilized by Dr. JohnLambris and colleagues (University of Pennsylvania) to calculate IC₅₀values of Compstatin and its analogs (Mallik et al., 2005. J. Med. Chem.48, 274-286). Complement activation inhibition is assessed by measuringthe inhibition of serum C4 fixation to ovalbumin-anti-ovalbumincomplexes in NHS. Microtiter wells are coated with ovalbumin (10 mg/ml).Wells are then saturated with BSA (10 mg/ml) for one hour at roomtemperature and a 1:2,000 dilution of rabbit anti-ovalbumin antibodyadded to form immunocomplexes by which complement can be activated.Peptides at various concentrations are then added directly to each well,followed by a 1:80 dilution of NHS in GVB++. After a 30 minuteincubation, bound C4 is detected using a 1:2,000 dilution of a goatanti-C4 antibody, followed by a 1:2,500 dilution of donkey anti-goat HRPsecondary antibody. The plates are then developed with tetramethylbenzindine, the reactions terminated with H₂SO₄, and absorbance read at450 nm. Percent inhibition is normalized by considering 100% activationequal to activation occurring in the absence of peptide.Heat-inactivated NHS is utilized as a negative control for activation.NHS+CP1 are used as a control of inhibition of activation.

IC₅₀ values for the selected peptide compounds are determined byplotting the percent inhibition against peptide concentration. CPinhibits C4 activation via the classical and lectin pathways, and CP hasnominal effects on activation of the alternative pathway (Bonaparte etal., 2008. J. Virol. 82, 817-827, Hair et al., 2010. Molec. Immunol. 47,792-798). Using CP1 as the benchmark, the relative inhibitory activitiesfor all peptide compounds are thus directly determined.

The IC₅₀ values for the CP peptides in normal rat sera (NRS) aredetermined. Wild-type CP and CP1 have been demonstrated to suppressantibody-initiated complement activation in NRS (Hair et al., 2010.Molec. Immunol. 47, 792-798). Determination of the IC₅₀ values for thepeptide compounds in NRS are critical for dose-ranging experiments inrats.

Other aspects, modifications, and embodiments are within the scope ofthe following claims.

REFERENCES

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The invention claimed is:
 1. A method of reducing activity of theclassical pathway of the complement system in a subject with a diseaseassociated with complement-mediated tissue damage comprisingadministering to the subject in need thereof a pharmaceuticalcomposition comprising a synthetic peptide comprising the amino acidsequence of SEQ ID NO: 9, wherein the subject has complement-mediatedtissue damage.
 2. The method of claim 1, wherein the peptide comprisesan N-terminal acetylation.
 3. The method of claim 1, wherein thepharmaceutical composition comprises at least one pharmaceuticallyacceptable carrier, diluent, or excipient.
 4. The method of claim 2,wherein the pharmaceutical composition comprises at least onepharmaceutically acceptable carrier, diluent, or excipient.
 5. Themethod of claim 1, wherein the disease is selected from the groupconsisting of systemic lupus erythematosus (SLE), autoimmune hemolyticanemia (AIHA), ischemia-reperfusion injury (IRI), rheumatoid arthritis,multiple sclerosis, myasthenia gravis, membranoproliferativeglomerulonephritis, serum sickness, Adult Respiratory Distress Syndrome(ASDS), stroke, myocardial infarction, allo- or xeno-transplantationinjury, hyperacute rejection, graft versus host disease (GVHD),Alzheimer's disease, burn injuries, hemodialysis damage, cardiopulmonarybypass damage, paroxysmal nocturnal hemoglobinuria (PNH), and hereditaryangioedema (HAE).
 6. The method of claim 5, wherein the disease issystemic lupus erythematosus (SLE).
 7. The method of claim 5, whereinthe disease is autoimmune hemolytic anemia (AIHA).
 8. The method ofclaim 5, wherein the disease is ischemia-reperfusion injury (IRI). 9.The method of claim 2, wherein the disease is selected from the groupconsisting of systemic lupus erythematosus (SLE), autoimmune hemolyticanemia (AIHA), ischemia-reperfusion injury (IRI), rheumatoid arthritis,multiple sclerosis, myasthenia gravis, membranoproliferativeglomerulonephritis, serum sickness, Adult Respiratory Distress Syndrome(ASDS), stroke, myocardial infarction, allo- or xeno-transplantationinjury, hyperacute rejection, graft versus host disease (GVHD),Alzheimer's disease, burn injuries, hemodialysis damage, cardiopulmonarybypass damage, paroxysmal nocturnal hemoglobinuria (PNH), and hereditaryangioedema (HAE).
 10. The method of claim 9, wherein the disease issystemic lupus erythematosus (SLE).
 11. A method of reducing activity ofthe classical pathway of the complement system in a subject with adisease associated with complement-mediated tissue damage comprisingadministering to the subject in need thereof a pharmaceuticalcomposition comprising a synthetic peptide comprising at least about 90%sequence identity to the amino acid sequence of SEQ ID NO: 9, whereinthe subject has complement-mediated tissue damage.
 12. The method ofclaim 11, wherein the peptide comprises an N-terminal acetylation. 13.The method of claim 11, wherein the pharmaceutical composition comprisesat least one pharmaceutically acceptable carrier, diluent, or excipient.14. The method of claim 12, wherein the pharmaceutical compositioncomprises at least one pharmaceutically acceptable carrier, diluent, orexcipient.
 15. The method of claim 11, wherein the disease is selectedfrom the group consisting of systemic lupus erythematosus (SLE),autoimmune hemolytic anemia (AIHA), ischemia-reperfusion injury (IRI),rheumatoid arthritis, multiple sclerosis, myasthenia gravis,membranoproliferative glomerulonephritis, serum sickness, AdultRespiratory Distress Syndrome (ASDS), stroke, myocardial infarction,allo- or xeno-transplantation injury, hyperacute rejection, graft versushost disease (GVHD), Alzheimer's disease, burn injuries, hemodialysisdamage, cardiopulmonary bypass damage, paroxysmal nocturnalhemoglobinuria (PNH), and hereditary angioedema (HAE).
 16. The method ofclaim 15, wherein the disease is lupus erythematosus (SLE).
 17. Themethod of claim 15, wherein the disease is autoimmune hemolytic anemia(AIHA).
 18. The method of claim 15, wherein the disease isischemia-reperfusion injury (IRI).
 19. The method of claim 12, whereinthe disease is selected from the group consisting of systemic lupuserythematosus (SLE), autoimmune hemolytic anemia (AIHA),ischemia-reperfusion injury (IRI), rheumatoid arthritis, multiplesclerosis, myasthenia gravis, membranoproliferative glomerulonephritis,serum sickness, Adult Respiratory Distress Syndrome (ASDS), stroke,myocardial infarction, allo- or xeno-transplantation injury, hyperacuterejection, graft versus host disease (GVHD), Alzheimer's disease, burninjuries, hemodialysis damage, cardiopulmonary bypass damage, paroxysmalnocturnal hemoglobinuria (PNH), and hereditary angioedema (HAE).
 20. Themethod of claim 19, wherein the disease is systemic lupus erythematosus(SLE).
 21. A method of reducing activity of the classical pathway of thecomplement system in a subject with a disease associated withcomplement-mediated tissue damage comprising administering to thesubject in need thereof a pharmaceutical composition comprising asynthetic peptide comprising the amino acid sequence of SEQ ID NO: 9,optionally with one or two conservative amino acid substitutions,wherein the subject has complement-mediated tissue damage.
 22. Themethod of claim 21, wherein the peptide comprises an N-terminalacetylation.
 23. The method of claim 21, wherein the pharmaceuticalcomposition comprises at least one pharmaceutically acceptable carrier,diluent, or excipient.
 24. The method of claim 22, wherein thepharmaceutical composition comprises at least one pharmaceuticallyacceptable carrier, diluent, or excipient.
 25. The method of claim 21,wherein the disease is selected from the group consisting of systemiclupus erythematosus (SLE), autoimmune hemolytic anemia (AIHA),ischemia-reperfusion injury (IRI), rheumatoid arthritis, multiplesclerosis, myasthenia gravis, membranoproliferative glomerulonephritis,serum sickness, Adult Respiratory Distress Syndrome (ASDS), stroke,myocardial infarction, allo- or xeno-transplantation injury, hyperacuterejection, graft versus host disease (GVHD), Alzheimer's disease, burninjuries, hemodialysis damage, cardiopulmonary bypass damage, paroxysmalnocturnal hemoglobinuria (PNH), and hereditary angioedema (HAE).
 26. Themethod of claim 25, wherein the disease is systemic lupus erythematosus(SLE).
 27. The method of claim 25, wherein the disease is autoimmunehemolytic anemia (AIHA).
 28. The method of claim 25, wherein the diseaseis ischemia-reperfusion injury (IRI).
 29. The method of claim 22,wherein the disease is selected from the group consisting of systemiclupus erythematosus (SLE), autoimmune hemolytic anemia (AIHA),ischemia-reperfusion injury (IRI), rheumatoid arthritis, multiplesclerosis, myasthenia gravis, membranoproliferative glomerulonephritis,serum sickness, Adult Respiratory Distress Syndrome (ASDS), stroke,myocardial infarction, allo- or xeno-transplantation injury, hyperacuterejection, graft versus host disease (GVHD), Alzheimer's disease, burninjuries, hemodialysis damage, cardiopulmonary bypass damage, paroxysmalnocturnal hemoglobinuria (PNH), and hereditary angioedema (HAE).
 30. Themethod of claim 29, wherein the disease is systemic lupus erythematosus(SLE).